BspQI
BspQI waxa lagu soo celin karaa E. coli oo aqoonsan kara goobo gaar ah oo lagu soo saaro hoos
BspQI, IIs restriction endonuclease restriction endonuclease, waxa ay ka soo jeedaan dib-u-habayn E. coli cadaadis kaas oo sidda gen BspQI ee cloned oo wax laga beddelay oo ka yimid Bacillus sphaericus.Waxay aqoonsan kartaa goobo gaar ah, iyo taxanaha aqoonsiga iyo goobuhu waa sida soo socota:
5' · · · · GCTCTTC(N) · · · · · · · · · · 3'
3' · · · · CGAGAAG(NNNN) · · · · 5'
Tilmaamaha Alaabta
1. Dhaqdhaqaaq sare, dheefshiidka degdega ah;
2. Dhaqdhaqaaqa xiddiga oo hooseeya, hubinta goynta saxda ah sida "scalpel";
3. BSA la'aanteed iyo xoolaha asalkoodu bilaash yahay;
Dareenka Methylation
Dwaxaan ahay methylation:Aan Xasaasi ahayn;
Dcm methylation:Aan Xasaasi ahayn;
Methylation CpG:Aan Xasaasi ahayn;
Xaaladaha kaydinta
Alaabta waa in lagu raro ≤ 0℃;Ku kaydi heerkulka -25 ~ 15 ℃
Kaydinta kaydinta
20mM Tris-HCl, 0.1mM EDTA, 500 mM KCl, 1.0 mM dithiothreitol, 500 µg/ml Recombinant Albumin, 0. 1% Trition X- 100 iyo 50% glycerol (pH 7.0 @ 25°C).
Qeexitaan Unug
Hal unug ayaa lagu qeexay cadadka insaymiska loo baahan yahay si loo dheefshiido 1µg ee kontoroolka gudaha DNA 1 saac 50°C wadarta mugga falcelinta ee 50 µL.
Xakamaynta tayada
Qiimaynta Nadiifinta Barootiinka (SDS-PAGE):Nadiifinta BspQI waxaa ≥95% lagu go'aamiyay falanqaynta SDS-PAGE.
RNase:10U ee BspQI oo leh 1.6μg MS2 RNA muddo 4 saacadood ah oo ah 50℃ ma keenayso hoos u dhac sida ay go'aamisay agarose gel electrophoresis.
Waxqabadka DNA-ga Aan Gaarka ahayn:10U of BspQI oo leh 1μg λ DNA at 50℃ 16 saacadood, marka la barbar dhigo 50 ℃ 1 saac, ma keeno DNA xad dhaaf ah sida lagu go'aamiyay agarose gel electrophoresis.
Dib u dhigista iyo dib u dhigista:Ka dib dheefshiidka 1 μg λDNA oo leh 10U BspQI, jajabyada DNA waxaa lagu dhejin karaa T4 DNA ligase at 16ºC.Oo jajabyadan isku xidhan waxa lagu gooyn karaa BspQI.
E. koli DNA: E.coli 16s rDNA-gaar ah TaqMan qPCR ogaanshaha waxay muujisay in E.coli genome hadhaaga ≤ 0.1pg/ul.
Hadhaaga borotiinka martida loo yahay:≤50 ppm
Bakteeriya Endotoxin: LAL-tijaabo, marka loo eego daabacaadda Chinese Pharmacopoeia IV 2020, habka tijaabada xadka jel, qaanuunka guud (1143).Bakteeriyada endotoxin waa inay noqotaa ≤10 EU/mg.
Nidaamka falcelinta iyo xaaladaha
| Qayb | Mugga |
| BspQ I (10 U/μL) | 1 μL |
| DNA | 1 μg |
| 10 x BspQ I Buffer | 5 μL |
| dd H2O | Ilaa 50 μl |
Xaaladaha falcelinta: 50 ℃, 1 ~ 16 saac
Dhaqdhaqaaq la'aanta kulaylka:80°C 20 daqiiqo
Nidaamka falcelinta lagu taliyay iyo xaaladaha ayaa ku siin kara saameyn dheefshiidka enzyme xad wanaagsan, taas oo loogu talagalay tixraac kaliya, fadlan tixraac natiijooyinka tijaabada ah faahfaahinta.
Codsiga alaabta
Xannibaad dheefshiidka endonuclease, cloning degdeg ah.
Xusuusin
1. Mugga enzyme ≤ 1/10 ee mugga falcelinta.
2. Dhaqdhaqaaqa xiddiguhu wuxuu dhici karaa marka fiirsashada glycerol ay ka badan tahay 5%.
3. Dhaqdhaqaaqa jeexjeexa ayaa dhici kara marka substrate ka hooseeyo saamiga lagu taliyey.
